Acanthamoeba, a genus of small free-living amebas, includes isolates obtained from human infections, isolates obtained from nature that are pathogenic to animal models, and isolates that apparently are nonpathogenic. The species A. castellanii includes all three types of strains. Classification of Acanthamoeba is difficult at the species level and little is known about intraspecific relationships of pathogenic and nonpathogenic forms. This project addresses the problem, using variations in mitochondrial DNA (mtDNA) as a basis for investigating possible distinctions between potential pathogens and nonpathogens. Four basic approaches are proposed: (1) A survey of mtDNA fragment patterns obtained by electrophoresis of restriction endonuclease digests from independent isolates of A. castellanii; (2) Quantitative DNA/DNA filter hybridization of total genome mtDNA from the various isolates; (3) Qualitative DNA/DNA hybridization using restriction fragments, electrophoresis, and Southern transfers to identify homologous sequences; and (4) Comparative physical (DNA fragment) mapping of mitochondrial genomes from representative isolates.